Single base mutations play a role in cancer and other human diseases, and can be useful markers for diagnostic tests. Mutations in viral genomes can cause resistance to antiviral drugs. Unfortunately, clinical samples often contain low-levels of mutant cells and viruses mixed with a vast excess of wild-type cells or viruses. Development of sensitive and accurate methods for detection of point mutations is an important challenge.
Single base mutations can be detected with restriction fragment length polymorphism/Southern blot assays (Vary, C. P. et al. Genet Anal 13, 59-65 (1996); Chen, J. & Viola, M. V. Anal Biochem 195, 51-6 (1991).), oligonucleotide ligation assays (Redston, M. S., Papadopoulos, N., Caldas, C., Kinzler, K. W. & Kern, S. E. Gastroenterology 108, 383-92 (1995); and Rothschild, C. B., Brewer, C. S., Loggie, B., Beard, G. A. & Triscott, M. X. J Immunol Methods 206, 11-9 (1997)), and allele-specific PCR (AS-PCR) assays or the amplification refractory mutation system (Clayton, S. J. et al. K, Clin Chem 46, 1929-38 (2000); Takeda, S., Ichii, S. & Nakamura, Y. Hum Mutat 2, 112-7 (1993); Germer, S., Holland, M. J. & Higuchi, R. Genome Res 10, 258-66 (2000); and Kwok, S. et al. Nucleic Acids Res 18, 999-1005 (1990)). The limit of detection of those assays is often limited to 0.1-1% (mutant/wild-type ratio of 1/1,000-1/100). Other assays can detect single base mutations in unique sequence contexts at lower levels (McKinzie, P. B. & Parsons, B. L. Mutat Res 517, 209-20 (2002); Lichtenstein, A. V., Serdjuk, O. I., Sukhova, T. I., Melkonyan, H. S. & Umansky, S. R. Nucleic Acids Res 29, E90-0 (2001); Schimanski, C. C., Linnemann, U. & Berger, M. R. Cancer Res 59, 5169-75 (1999); and Kaur, M. et al. Mutagenesis 17, 365-74 (2002)), but cannot be generally applied. Detection of point mutations with real-time quantitative PCR (Q-PCR) assays is often hampered by cross-hybridization of probes to wild-type templates (Oliver, D. H., Thompson, R. E., Griffin, C. A. & Eshleman, J. R. J Mol Diagn 2, 202-8 (2000)).
New initiatives support development of assays for early diagnosis of common cancers, for families at increased risk and population screening (Srivastava, S. & Rossi, S. C. Int J Cancer 69, 35-7 (1996); Sidransky, D. et al. Science 256, 102-5 (1992); and Traverso, G. et al. N Engl J Med 346, 311-20 (2002)). Such assays may also help monitor bone marrow transplant engraftment, and disease recurrence in cancer patients after treatment. Pancreatic cancer is usually diagnosed at an advanced stage, and early diagnosis is critical for improving survival rates. KRAS2 mutations are present in most pancreatic cancers and can be detected in pancreatic duct juice, as well as plasma and stool (Wilentz, R. E. et al. Cancer 82, 96-103 (1998); and Mulcahy, H. & Farthing, M. J. Ann Oncol 10 Suppl 4, 114-7 (1999)). However, KRAS2 mutations can also be detected in pancreatic duct juice and stool from patients with chronic pancreatitis or pancreatic intraepithelial neoplasias (PanINs) (Hruban, R. H., Wilentz, R. E. & Kern, S. E. Am J Pathol 156, 1821-5 (2000) and Caldas, C. et al. Cancer Res 54, 3568-73 (1994)). Qualitative detection of mutant KRAS2 alone is not an accurate predictor of pancreatic cancer (Watanabe, H. et al. Pancreas 17, 341-7 (1998)), but there is a need in the art for quantitative assays for KRAS2 mutations in biological fluids might be able to distinguish between pancreatic cancer and other conditions (Tada, M. et al. Dig Dis Sci 43, 15-20 (1998)).
In the management of patients with viral infections, for example, HIV, antiretroviral drugs can select for HIV-1 with drug-resistance mutations in protease and reverse transcriptase. Most HIV-1 genotyping assays are relatively insensitive for detection of minority variants with resistance mutations. A recent study suggests that the presence of such variants can influence treatment response (Mellors J., et al, Antiviral Therapy., 8: S150, 2003). A multicenter study used different assays to detect the K103N drug resistance mutation (Halvas E., et al, Antiviral Therapy, 8, S102, 2003). Thus, there is also a need in the art for assays to detect point mutations to aid in the management of patients viral infections, for example, HIV infections.
Several assays can detect single base substitutions, but their limit of detection is generally 0.1-10% (ratio of mutant/wild-type, equivalent to 1/10-1/1,000). DNA sequencing typically can detect mutant molecules only at a 10-25% level, depending in part on sequence context [Larder, 1993 #40]. Restriction fragment length polymorphism (RFLP)/Southern blot assays typically have a limit of detection of 0.5-5% [Vary, 1996 #3][Chen, 1991 #19]. The oligonucleotide ligation assay (OLA), even with sensitive capillary electrophoresis detection, can only detect minor species at about 0.1-1% [Redston, 1995 #4][Rothschild, 1997 #5]. Ligation chain reaction uses four oligonucleotides and ligase as an alternative to polymerase, but its limit of detection is approximately 0.1 to 1% [Barany, 1991 #49]. Allele-specific PCR (AS-PCR) or the amplification refractory mutation system (ARMS) can only detect approximately 1% of mutant DNA [Germer, 2000 #44; Kwok, 1990 #28] since the Taq polymerase still extends to some degree from a mispaired primer. In addition, most of these techniques lack of accurate quantification. Real-time quantitative PCR (Q-PCR) is remarkably quantitative over a wide dynamic range, but has been difficult to apply to the detection of point mutations because mutant probes that differ by a single base often cross-hybridize with the wild-type template [Oliver, 2000 #43]. Thus, there is a need in the art for assays with increased sensitivity to detect, for example, a single copy of a virus in a sample.